We are designing the methods for absolute quantification, developing and testing standards of tagged proteins (in collaboration with Dr. Iain Fraser) and designing peptides for AQUA method of MS-based quantification. The experimental model is the osteoclast development from macrophages. Understanding the mechanisms of osteoclast formation and action is crucial for the progress in the studies of rheumatoid arthritis and osteoporosis. We use well characterized murine macrophage RAW 264.7 cell line. The cells fuse to form multinucleated osteoclasts when stimulated with receptor activator of nuclear factor kappa B ligand (RANKL), but the differentiation process is inhibited by sphingosine-1 -phosphate (S1P). There are changes in protein expression connected with macrophage differentiation into osteoclasts. The mRNA levels of many proteins change and we want to see if these changes are reflected in the changes of the cell proteome. We have optimized the cell culture conditions and osteoclast enrichment. Using SILAC (stable isotope labeling with amino acids in cell culture) we compared the proteomes of untreated RAW 264.7 macrophages, differentiated osteoclasts. We are doing the same for macrophages treated with RANKL/S1P combination. The analysis revealed a set of differentially expressed proteins, which we used to design a set of standard peptides for absolute quantification by mass spectrometry. We obtained a library of 409 standard, synthetic peptides which we are using to assess the protein expressions in macrophages and osteoclasts. The Multiple Reaction Monitoring (MRM) analysis of a narrower set of chosen peptides differentially expressed under different experimental conditions will provide absolute numbers of molecules. These will be used for mathematical modeling of cellular pathways involved in osteoclast development. We are using the MRM approach in the collaboration with Dr. Rajat Varma on the exploration of the commonality of gamma chain in interleukin receptors. We have designed and received a set of 77 T-cell signaling-specific peptides and are testing them for use in this project.